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Evolutionary Stability of Ubiquitous Root Symbiosis

Periodic Reporting for period 4 - HeteroDynamic (Evolutionary Stability of Ubiquitous Root Symbiosis)

Reporting period: 2020-10-01 to 2022-03-31

In the HeteroDynamic research program we address the evolutionary stability of AM symbiosis, and base our work on the conceptual hypothesis that the symbiosis is stable thanks to a unique genomic organization, a temporally dynamic heterokaryosis, in the fungal partner. This hypothesis is based on the assumptions that 1) heterokaryosis allows for 2) functional variation 3) over time - and my team works from different angles to test these.

Firstly we address heterokaryosis in AM fungi by developing a novel single nucleus genomics method for sequencing and assembly of AM fungal genomes from single nuclei. Reads from individual nuclei can then be mapped back to the assembly to evaluate sequence variance among nuclei from individual strains and even from within single spores. The physical sorting of nuclei using FACS had been established for one species prior to the onset of the HeteoDynamic research program. During the project the method has been developed to accommodate a range of species and a custom-made assembly workflow has been developed to handle the generated data. The method was published in January 2020 (Montoliu-Nerin et al 2020). An added value of this method is that it allows us to assemble genomes from a broad range of AM fungi that could previously not be sequenced due to challenges obtaining sufficient amount and purity of biological material from most AM fungi. Analysis of within strain genetic variation reveales overall low levels of polymorphism and the single nucleotide level. Based on these studies and work on repetitive DNA content in AM fungi the first PhD student defended her thesis in Dec 2020. Low levels of heterokaryosis suggest that the conceptual hypothesis of the HeteroDynamic project should be rejected. However, our comprehensive genome analysis has revealed interesting patterns of rDNA polymorphism, gene copy number variation and possibly variation in ploidy as means of maintaining genetic variation in AM fungal genomes. Through out the project we have continued to analyse these patterns to the extent it is possible with amplified genome DNA . A new PhD student (David) was recruited in December 2019 and has has since focused on verifying genetic polymorphism and examining signals of selection in the existing polymorphism. With the novel workflow for genome sequencing that we have established we have expanded the phylogenetic width of available genome data in Gloeromycota and we have publishing 21 new genomes from 12 genera across seven families.

Secondly, we set out to test for functional heterokaryosis, by simultaneously exposing fungal strains to different hosts. However, the growth experimental part of the HeteroDynamic project has not been as straightforward as the genomic part of the project and in the end the data from these experiments can not be used to test the initial hypothesis. The main reason being lack of fungal colonisation at the time of harvest. Despite this set back, the project has generated major additions of genomic data to the field of AM fungal genomics and ongoing analysis of these resources is building towards a solid understanding of the evolutionary history of these major plant symbionts. Our research is not only important for better understanding evolutionary dynamics of ancient mutualistic interactions between fungi and plants but are potentially important for future agriculture including the use of AM inoculum, optimized rotations and breeding for beneficial AM fungi. While negative feedback between fungi and host plants is important in maintaining high biodiversity in natural ecosystems its role in agriculture has not yet been tested and our studies build the foundation for such studies. To take advantage of growth promoting AM fungal communities in sustainable intensification of agriculture, systems need to be developed where crop rotations are tested for their ability to promote continually beneficial communities.
Over the course of the Heterodynamic project the Team has comprised of the PI, 2 post docs, 3 PhD students part time lab technical support, green house technician and part time bioinformatic support. At the time of the mid-term report all recruitments had been done. Post doc 2 left the project early because she secured a permanent position at SLU in Uppsala. To compensate for her a third PhD student was recruited to the team. He is set to finish his thesis in 2024. The project was prolonged for 1 year due to parental leaves in the team and delays due to covid. I am very thankful for this prolongation that has been absolutely essential for us to be able to get as far as we have with wrapping up the project.

At large the project has been executed according to DoA but in the end I can conclude that the the growth experiments designed to adress Hypothesis 3 and to build towards the conceptual hypothesis did not work out as planed. This means that much of the work performed by Post doc 1 and PhD 1 will not generate publications. These studies are currently included as a manuscript in the thesis of PhD 1 who is defending on 31 May, 2022. It is thus fortunate that the massive method development that we conducted in order to build reliable genome data for testing of Hypothesis 1 has generated spin off results that may in the end be the most important outcome of this project. We have taken full advantage of this method development and dramatically expanded the taxonomic range of genome data available for AM fungi (Fig 1). Our workflow has presented a pathway for generating genome data from the eukaryotic majority that we know only as single celled organisms from diverse lineages. Despite some initial skepsisim towards this novel workflow, Im confident that it will Ian acceptance as we continue to demonstrate the stability of the generated data.

PhD 3 was recruited to replace Post doc 2 and thus has taken on the project originally designed for Post doc 2. He will continue work on adress Hypothesis 1 and 2 in his PhD, scheduled to be finished in 2024, thus not presented yet in this reprot.
In the Heterodynamic project a novel workflow for single nuclei sorting, sequencing and genome assembly was established and published (Montoliu-Nerin et al 2020). With this workflow we proceeded to generate data from 21 taxa representing seven families in Glomeromycota and published the most taxon rich phylogenetic analysis of Glomeromycota to data (Montoliu-Nerin et al 2021). Prior to our work, genome data was only available from a handfull of species that could be grown under axenic culture conditions or formed larger soporcarps that can be sampled for metagenomic approaches (Fig 1). In the end our workflow for genom sequencing and assembly is the most important contribution from the Heterodynamic project and I think it will prove useful also in other non model micro-eukaryotes, which do represent the most eukaryoti, for which genome data was previously not available. A phylogenomic analysis based on this data is shortly ready for resubmission after revision and will constitute a major expansion for future genomic analysis in this important lineage of plant symbiotic fungi.
Available AM fungal genomes